PROJECT SUMMARY/ABSTRACT In this project, we will systematically define the genetic and cellular adaptations associated with an extremophile phenotype in bacteria ? extraordinary resistance to the effects of ionizing radiation (IR). Instead of studying prototypical IR resistant species such as Deinococcus radiodurans, we are generating IR resistant Escherichia coli populations by directed evolution. The resulting strains approach, and in some cases exceed the levels of IR resistance seen in Deinococcus. Analysis of the mutations underlying the acquired phenotype will allow us to quickly home in on the key cellular innovations. The ultimate goal is to define ALL processes and mechanisms that contribute to an extreme IR resistance phenotype. The directed evolution of this phenotype in E. coli provides a window that makes this possible. In this work we will both exploit and expand an existing resource of four highly evolved populations of E. coli. Using directed evolution, all of these have acquired high levels of IR resistance. The populations are designated IR-1-20, IR-2-20, IR-3-20, and IR-4-20. We have already defined the mutations most relevant to the phenotype in both IR-2-20 and IR-3-20. In addition, we are currently generating four new evolved populations from scratch, using a different type of radiation source, and further evolving the four existing populations. There are four specific aims: Aim 1 focuses on the evolution of new and existing populations, as well as definition of the mutations that make substantial contributions to the phenotype. Defined strains with key contributing mutations, isolated in an otherwise wild type background, will be constructed. Aim 2 represents a general effort to use the modern resources of systems biology to thoroughly characterize the evolved populations and single colony isolates derived from them. Aim 3 will focus on an exploration of one particular contributing mechanism of IR resistance involving genetic alterations in genes encoding proteins involved in replication restart. Aim 4 is the capstone. We will use information gained from aims 1-3 to transfer the IR resistance phenotype intact by introducing a defined set of mutations into Salmonella enterica. In this mentored research experience, Ms. Wolfsmith will be involved in work described under aim 2. We now have four highly evolved populations that are nearly as resistant to IR as is Deinococcus. The sheer numbers of mutations present provide a challenge in determining which of them is important for the IR resistance phenotype. We can narrow things down by comparing different populations and focusing on patterns found in two or more of them. However, that still leaves us with many dozens of prominent candidate mutations. We have constructed large numbers of strains in which particular candidate mutations are isolated in otherwise wild type backgrounds. We also have strains that combine some of these. All of these strains must be tested for growth rates, resistance to DNA damaging agents, and IR resistance. We will also be using mass spec and NMR to determine what effects the mutations have on the strain?s proteome and metabalome. Ms Wolfsmith will participate in all of these studies. In the process, she will gain experience with quite a number of different methods from basic genetics to the analysis of proteomic and metabalomic datasets. In addition to basic laboratory skills, Ms Wolfsmith will be asked to present one group meeting on her work near the end of the summer. She will also write a report on the project. As part of the team, Ms Wolfsmith will participate in weekly planning meetings led by me or one of the postdocs heading up this project (Steven Bruckbauer and Takeshi Shinohara). She will be expected to contribute to ideas about project directions and experiment planning. The supplement will add value to the student?s training, as the summer period provides time that is not available during the semester. More elaborate experiments can be planned and carried out. Learning can occur in the lab without distractions from other academic pursuits. All of the students will be relying on each other in a kind of cross-mentoring situation. I note that I typically have 8 undergraduate researchers working in the laboratory as a steady state and have trained over 70 in my time at the University of Wisconsin-Madison. These students are very successful after graduation. Recent graduates have entered graduate programs at Harvard, University of Washington, Stanford, Duke, and the University of Michigan, as well as MD/PhD programs at Washington University and University of Pennsylvania.